IL-1β induced down-regulation of miR-146a-5p promoted pyroptosis and apoptosis of corneal epithelial cell in dry eye disease through targeting STAT3

Aim To elaborate the underlying mechanisms by which IL-1β promote progression of Dry eye disease(DED) through effect on pyroptosis and apoptosis of corneal epithelial cells(CECs). Methods 400 mOsM solutions were used to establish the DED model (hCECs- DED). RT-qPCR was performed to measure IL-1β mRNA and miR-146a-5p in CECs. Western blotting was performed to measure STAT3, GSDMD, NLRP3, and Caspase-1 levels. Cell counting kit-8 assay was adopted to check cell viability. Apoptosis was detected by flow cytometry. ELISAs were performed to determine IL-18, IL-33 and LDH. The luciferase test detects targeting relationships. Results After treatment with 400 mOsM solution, cell viability decreased and apoptosis increased. Compared with hCECs, IL-1β was increased and miR-146a-5p was decreased in hCECs-DED. At the same time, GSDMD, NLRP3, Caspase-1, IL-18, IL-33 and LDH were significantly higher in hCECs-DED than in hCECs, while IL-1β silencing reversed this effect. In addition, IL-1β negatively regulated miR-146a-5p. MiR-146a-5p mimics eliminated the inhibition of hCECs-DED pyroptosis and apoptosis caused by IL-1β silencing. At the same time, miR-146a-5p reduced STAT3 levels in hCECs. Conclusion Highly expressed IL-1β promoted pyroptosis and apoptosis of hCECs- DED through downregulated miR-146a-5p and inhibited STAT3. Supplementary Information The online version contains supplementary material available at 10.1186/s12886-024-03396-8.


Introduction
Dry eye disease (DED) is very common and widespread around the world, with a prevalence of up to 50% [1,2].Clinical manifestations of DED are persistent irritation, and can be subdivided into aqueous-deficient DED and hyperevaporative DED.If untreated, it can lead to inflammatory damage to the cornea or conjunctiva, which can have an adverse impact on patients' quality of life and work productivity.Due to damage to corneal epithelial cells (CECs), it is one of the most important events in the progression of DED [3], and there was a study found that pyroptosis of CECs plays a pivotal role in DED [4].Therefore, the aim of this paper is to investigate the mechanism of pyroptosis of CECs in dry eyes.
DED is associated with inflammation of the ocular surface [5].Studies have found that levels of inflammatory cytokines are mostly elevated in patients with DED [6].As one of the most important pro-inflammatory cytokines, the role of interleukin-1 beta (IL-1β) in DED has attracted much attention of researchers [7,8].There was a report indicated that inhibiting IL-1β relieves DED by suppresses cular surface injury [9].While the mechanism of IL-1β promote the progress of DED is not clear.
miRNAs are small noncoding RNAs that regulate various physiological events or diseases [10].First discovered in the 1990s, the function of miRNAs in DED has been explored [11].Among them, miR-146a-5p (previously miR-146a) kindled our interest.It is in the second exon of the human LOC285628 gene.It suppresses the immune system and inflammation.It has also been shown to be associated with the pathogenesis of DED [12].Nowadays, increasing evidences indicated that the expression of some miRNAs can be regulated by IL-1β [13].Furthermore, miR-146a-5p is also an IL-1β-responsive miRNA [14].Here, we studied the function of IL-1β and miR-146a-5p in DED, and the underlying mechanism by which they affect DED.
In order to provides a potential therapeutic target for DED, in this study, the mechanism that IL-1β regulate pyroptosis and apoptosis of CECs through miR-146a-5p/ STAT3 axis were explored.

Cell treatment
Human corneal epithelial cell line (hCECs, #6510, Sciencell, USA) from ATCC was maintained in DMEM with 10% FBS, EGF, insulin, and antibiotics at 37 °C.Hyperosmotic treatment was used to establish hyperosmotic DED model in vitro.In brief, NaCl was used to prepare 400 mOsM solution, then hCECs were treated for 24 h as hCECs-DED).At the same time, 312 mOsM NaCl was used as the control group (hCECs group).

R G C G C A T T A C C A C G A A C T C C A T T C A; U6 F: C T T G C A T C C G C A T C A G A, R A A T G C A T C A T G A A G T T C C G A.
The internal controls were GADPH and U6.Gene fold change was calculated by 2 −ΔΔCt [15].

Cell counting kit-8 assay
Cell counting kit-8 assay (CCK-8, Takara Bio, China) was used to measure cell growth [17].Cells were cultured in 96-well plates, CCK-8 (10µL) was provided to each well and kept for 2 h incubation, and OD450 was read with a microplate reader.

Apoptosis analysis
Apoptosis was detected using flow cytometry.After treatment of cells according to different groupings, cells were stained with the Annexin V/FITC Kit (BD Biosciences, USA) according to the kit instructions.

Statistical analysis
Experiments in this study were independently performed 3 times, and data was shown as means ± SD.Data was analyzed by SPSS 22.0, and figure was mapped by using GraphPad Prism 7. Differences were analyzed by Student's t test or one-way ANOVA.P < 0.05 was defined statistically significant.

DED causes pyroptosis of hCECs
To establish the CEC DED model, hCECs were treated with 400 mOsM solutions and then labeled hCECs-DED.RT-qPCR results showed that compared with hCECs, expression IL-1β mRNA was increased in hCECs-DED (Fig. 1A), whereas miR-146a-5p were down-regulated in hCECs-DED (Fig. 1B).CCK-8 assay of cell viability revealed that treatment with 400 mOsM NaCl solution significantly inhibited cell viability (Fig. 1C).The same results were obtained by microscopic observation of cell numbers (Fig. 1D).Flow cytometry detection of apoptosis revealed a significant increase in apoptosis in the hCECs-DED group (Fig. 1E).Pyroptosis is characterized by Caspase-1-induced GSDMD-driven cell lysis and NLRP3 inflammasome activation.Therefore, we detected the pyroptosis-related proteins GSDMD, NRLP3 and Caspase-1 by Western blotting.The results showed that compared with the hCECs group, the expression of GSDMD, NRLP3 and Caspase-1 proteins in the hCECs-DED group was significantly increased (Fig. 1F).In addition, ELISA for IL-18, IL-33 and LDH concentrations showed a significant increase in IL-18 and IL-33 concentrations and LDH release in the hCECs-DED group (Fig. 1G).In summary, the above experiments showed that dry eyes increased the apoptosis of hCECs and promoted the expression of pyroptosis-related proteins.

si-IL-1β transfection inhibits pyroptosis and apoptosis of hCECs
To investigate whether si-IL-1β is associated with pyroptosis and apoptosis of hCECs, we transfected si-IL-1β in hCECs-DED.After transfected si-IL-1β into hCECs-DED, expression of IL-1β mRNA and protein were drastically declined in si-IL-1β group (Fig. 2A and B).Data also indicated that IL-1β silence elevated miR-146a-5p in hCECs-DED (Fig. 2C).Both CCK-8 and microscopic observation of cell numbers revealed significantly higher cell numbers in the si-IL-1β group than in the NC group (Fig. 2D and E).Cell apoptosis was detected by flow cytometry and the results showed that transfection with si-IL-1β significantly inhibited apoptosis of hCECs-DED (Fig. 2F).Similar results were obtained by Western blotting for pyroptosis-related proteins (Fig. 2G).Measurement of inflammatory factor (IL-18, IL-33) concentrations and LDH release revealed a decrease in inflammatory factor concentrations and relief of cellular damage (Fig. 2H).Therefore, the ability of IL-1β to affect pyroptosis and apoptosis of hCECs-DED.

Discussion
DED is a major ophthalmic disease worldwide, characterized by loss of tear film homeostasis leading to eye discomfort and even visual impairment, which seriously affects the quality of life of patients [19].A growing number of studies suggest that chronic immune processes play a key role in the pathogenesis of DED, characterized by elevated levels of pro-inflammatory cytokines and inflammation, which further leads to disruption of the corneal epithelial barrier [20].Tear hyperpermeability leads to symptoms of dry eye affecting epithelial cell function.The osmolality of normal subjects ranged from 290 to 330 mOsm / L, while the osmolality of dry eye patients ranged from 315 to 365 mOsm/L [21].Studies have shown that the use of 350-500 mOsM hypertonic medium to construct hCECs-DED [22].In our study, 400 mOsM hypertonic medium modeling effect is better.At the same time, this study found that high osmotic pressure can cause pyroptosis and apoptosis of hCECs (Fig. 1).
Pyroptosis is also known as cellular inflammatory necrosis and is a programmed cell death mediated by caspase-1.Caspase-1 is instrumental in the process of transforming two proinflammatory cytokines, IL-1β and IL-18, into mature forms within the inflammasome pathway.Caspase-1 also cleaves the pore-forming protein GSDMD into its active form N-GSDMD, which triggers a cascade of events that culminates in plasma membrane rupture and intracellular content release, resulting in a localized or systemic inflammatory response [23].IL-1β is a key mediator of the inflammatory response [24].It is recognized that inflammation such as high levels of IL-1β, is an underlying cause of DED.On the other hand, pyroptosis also leads to an increase in the expression of IL-1β [25,26].There were many researches demonstrated that IL-1β was up-regulated in DED patients [9,27,28].In addition to pyroptosis, caspase-1 is also involved in apoptosis in inflammatory situations [29].Our results showed that IL-1β was highly expressed in DED model hCECs, and IL-1β silencing inhibited the thermal degeneration of DED model hCECs (Fig. 2).Therefore, high level of IL-1β in DED patients promoted pyroptosis and apoptosis of hCECs, and then pyroptosis hCECs released pro-inflammatory cytokines that further worsen the condition of DED.This process formed an "inflammatory vicious cycle" in DED.
In the present study miR-146a-5p was also found to be significantly reduced in hCECs-DED.The use of miR-146a-5p mimics treatment can increase cell viability and inhibit the occurrence of inflammation (Fig. 3).
STAT3 was identified as a target gene of miR-146a-5p by bioinformatics analysis, while luciferase assay and protein blotting confirmed this [35].Signal transducer and STAT3 is a latent cytoplasmic protein associated with inflammation and cellular pyroptosis [36,37].One of the pathogenic mechanisms of Yorkren syndrome is STAT3-mediated epithelial cell dysfunction [38].In addition, recent studies have found that IL-1β regulates wound healing in the corneal epithelium via p16Ink4a-STAT3 signaling [39].STAT3 inhibitor ameliorates dry eye symptoms in mice [40].In this study, we found that miR-146a-5p significantly decreased STAT3 expression (Fig. 4).Overexpression of STAT3 significantly inhibited the effects of miR-146a-5p on the expression of inflammatory factors and the proliferation of hCECs and pyroptosis and apoptosis (Fig. 5).

Conclusion
In summary, this study found that IL-1β promotes pyroptosis and apoptosis in the DED model by downregulating miR-146a-5p and promoting STAT3.Our findings could provide a theoretical basis for the treatment of DED.